Replication-defective adenovirus human type 5 recombinant as a vaccine carrier

ABSTRACT

A replication defective recombinant adenovirus is provided which contains a complete deletion of its E1 gene and at least a partial deletion of its E3 gene, said virus containing in the site of the E1 deletion a sequence comprising a non-adenovirus promoter directing the replication and expression of DNA encoding a heterologous protein from a disease-causing agent, which, when administered to a mammal in said recombinant virus, elicits a substantially complete protective immune response against the agent. Pharmaceutical and veterinary products containing the recombinant adenovirus are provided.

CROSS-REFERENCE TO OTHER APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 08/973,233,filed Dec. 3, 1997, (now U.S. Pat. No. 6,019,978) which is a 35 USC §371application based on International Patent Application No.PCT/US96/09495, filed Jun. 5, 1996, which is a continuation of U.S.patent application Ser. No. 08/461,837, filed Jun. 5, 1995 (now U.S.Pat. No. 5,698,202) and claims the benefit of priority from U.S.provisional patent application No. 60/000,078, filed Jun. 8, 1995 (nowabandoned).

This invention was supported by the National Institutes of Health GrantNos. NIH Al 33683-02 and NIH AI 27435-05. The United States governmenthas certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to recombinant adenoviruses as vaccinecomponents, and more particularly, to the use of replication deficientadenoviruses as vaccine carriers, which induce protective immuneresponses in mammalian hosts.

BACKGROUND OF THE INVENTION

A replication competent, recombinant adenovirus (Ad) is an adenoviruswith intact or functional essential genes, (i.e., E1a, E1b, E2a, E2b andE4). Such recombinant viruses containing a variety of inserted geneshave been used as vaccine compositions with some success [see, e.g.Davis, U.S. Pat. No. 4,920,309].

One of these recombinant adenoviruses expressing the rabies G proteinwas shown to induce protective immunity in animals upon challenge withrabies virus [L. Prevac, J. Infect. Dis., 161:27-30 (1990)]. However,doses above 10⁶ plaque-forming units (pfu) of this replication-competentvirus were required to induce complete protection to viral challenge.Further, the use of these viruses in a live form capable of replicatingin vivo is an undesirable attribute of a vaccine component.

In contrast, adenoviruses which have been made replication deficient bydeletion of the Ad E1a and E1b genes have been used primarily for genetherapy protocols [See, e.g., Kozarsky and Wilson, Curr. Opin. Genet.Dev., 3:499-503 91993); Kozarsky et al, Som. Cell Mol. Genet.,19:449-458 (1993); see also, International Patent Application No.WO95/00655, published Jan. 5, 1995]. Such recombinant, replicationdeficient adenoviruses have been found to induce cell-mediated immuneresponses [Y. Yang et al, Proc. Natl. Acad. Sci. USA, 91:4407 (1994) andY. Yang et al, Immunity, 1:433-442 (August 1994)] and neutralizingantibodies [T. Smith et al, Gene Therapy, 5:397 (1993); K. Kozarsky etal, J. Biol. Chem., 269:13695 (1994)]. None of these articles relatingto the use of recombinant replication deficient Ad in gene therapy havemeasured the induction of a protective immune response.

Others have described the insertion of a foreign gene into areplication-defective adenovirus for putative use as a vaccine [See,e.g. T. Ragot et al, J. Gen. Virol., 74:501-507 (1993); M. Eliot et al,J. Gen. Virol., 71:2425-2431 (1990); and S. C. Jacobs et al, J. Virol.,66:2086-2095 (1992)]. Jacobs et al, cited above, describes a recombinantE1-deleted, E3 intact, Ad containing encephalitis virus protein Ns1under the control of a heterologous cytomegalovirus (CMV) promoter. Whenmice were immunized with the recombinant Ad vaccines and challenged withvirus, Jacobs et al obtained only partial protection (at most a 75%protection) for an average survival of 15 days. Eliot et al, citedabove, describe a recombinant E1-deleted, partially E3-deleted Ad withpseudorabies glycoprotein 50 inserted into the E1 deletion site underthe control of a homologous Ad promoter. In rabbits and mice, afterimmunization and challenge, only partial protection was obtained (i.e.,about one-third). Ragot et al, cited above, describe a recombinantE1-deleted, partially E3-deleted Ad with Epstein Barr virus glycoproteingp340/220 inserted into the E1 deletion site under the control of ahomologous Ad promoter. In marmosets (tamarins) after three high dose(5×10⁹ pfu, 1×10¹⁰ pfu and 2×10¹⁰ pfu), intramuscular immunizations andviral challenge, full protection was obtained.

For certain highly infectious diseases, such as rabies, there is ademand for an effective vaccine. Desirably, a vaccine should beeffective at a low dosage to control the occurrence of side effects orto enable sufficient amounts of vaccine to be introduced into animals inthe wild. Currently, a vaccinia rabies glycoprotein (VRG) vaccine isbeing used for oral wild-life immunization [B. Brochier et al, Vaccine,22:1368-1371 (1994)]. However, doses above 10⁶ pfu are required toinduce complete protection.

There thus remains a need in the art for a method of vaccinating againstvarious disease states, and particularly rabies, which is safe andhighly effective.

SUMMARY OF THE INVENTION

The inventors have surprisingly found compositions and methods ofvaccinating a human and/or animal against a disease using an adenovirusdefective vaccine composition, which produces a high level of protectionupon administration of a low vaccine dose. For example, vaccination witha vaccine composition described herein, which is directed againstrabies, has been found to require as little as a single dose of 10⁴ pfuof rabies vaccine vector to induce complete protection. This effect isalso accomplished by administration routes other than the oral route.

Thus, in one aspect, the invention provides a replication-defectiverecombinant adenovirus (rAd) vaccine containing DNA encoding a selectedheterologous protein from a disease-causing agent, which elicits aprotective immune response against the agent. This recombinantadenovirus of the invention contains at least a partial, but functional,deletion of the Ad E3 gene. Further in the site of the E1a/E1b deletionwhich renders the Ad replication-defective, the recombinant viruscontains a sequence comprising a non-adenovirus promoter directing thereplication and expression of the DNA encoding the heterologous protein.For example, an exemplary rAd is Adrab.gp, which contains a rabies gpgene and is useful in a method for treating or preventing rabies.

In another aspect, the invention provides pharmaceutical and veterinarycompositions which contain the rAd of the invention.

In still another aspect, the invention provides for the use of the rAdin the manufacture of the compositions described above.

In yet a further aspect, the invention provides a method of vaccinatinga human or animal against disease comprising administering to said humanor animal an effective amount of a replication-defective recombinantadenovirus vaccine containing DNA encoding a selected heterologousprotein which elicits a protective immune response against an agentcausing the disease. This adenovirus of the invention contains at leasta partial, but functional, deletion of the Ad E3 gene. Further in thesite of the E1a/E1b deletion which renders the Ad replication-defective,the recombinant virus contains a sequence comprising a non-adenoviruspromoter directing the replication and expression of the DNA encodingthe heterologous protein.

In another aspect, the present invention provides a method of preventingrabies infection in an animal comprising administering to the animal aneffective amount of a recombinant replication-defective Adrab.gpadenovirus containing DNA encoding a rabies virus glycoprotein.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the 1650 bp rabies glycoproteingene from Evelyn Rockitniki Abelseth strain excised from the pSG5.ragpplasmid by cleavage with BglII. The 1650 bp sequence spans nucleotide1178 to 2827 of SEQ ID NO: 1.

FIG. 1B is a schematic map of the pAd.CMVlacZ (also known asH5.020CMVlacZ) plasmid, which contains adenovirus map units (m.u.) 0-1as represented by the black bar at the top of the circular plasmid,followed by a cytomegalovirus enhancer/promoter (CMV enh/prom)represented by the striped arrow to the right of the black bar, a humanbetagalactosidase gene represented by the dark gray bar at the righthandside of the circular plasmid; a polyadenylation signal represented bythe short white bar at the bottom of the circular plasmid, adenovirusm.u. 9-16 represented by the long black bar at the lower lefthandportion of the circular plasmid and plasmid sequences from plasmidpAT153 including an origin of replication and ampicillin resistance generepresented by the light gray bar at the upper lefthand portion of thecircular plasmid. Restriction endonuclease enzymes are represented byconventional designations in this plasmid. NotI digestion removes theLacZ gene from this plasmid.

FIG. 1C is a schematic map of the plasmid pAdCMV.rabgp which resultsfrom blunt end cloning of the BglII fragment of pSG5.ragp to the largerNotI fragment of pAdCMV.lacZ. pAdCMV.rapgp is substantially similar tothe pAd.CMVlacZ plasmid, but which contains the rabies glycoproteinsequence in place of the lacZ gene. pAdCMV.rapgp [SEQ ID NO: 1] containsadenovirus m.u. 0-1 as represented by the black bar at the top of thecircular plasmid (nucleotides 12 to 364 of SEQ ID NO: 1); followed by acytomegalovirus enhancer/promoter (CMV enh/prom) represented by thestriped arrow to the right of the black bar [nucleotides 382 to 863 ofSEQ ID NO: 1]; a rabies glycoprotein gene represented by the dotted barat the righthand side of the circular plasmid (nucleotides 1178 to 2827of SEQ ID NO: 1); a polyadenylation signal represented by the shortwhite bar at the lower righthand portion of the circular plasmid[nucleotides 2836-3034 of SEQ ID NO: 1]; adenovirus m.u. 9-16represented by the long black bar at the lower portion of the circularplasmid (nucleotides 3061 to 5524 of SEQ ID NO: 1); and plasmidsequences from plasmid pAT153 including an origin of replication andampicillin resistance gene represented by the light gray bar at theupper lefthand portion of the circular plasmid (nucleotides 5525 to 8236of SEQ ID NO: 1). Restriction endonuclease enzymes are represented byconventional designations. SEQ ID NO: 2 provides the rabies proteinsequence encoded by the nucleotide sequence within pAdCMV.rabgp.

FIG. 1D is a schematic map of recombinant adenovirus Adrab.gp (alsoknown as H5.020CMV.rab), which results from homologous recombinationbetween pAdCMV.rabgp and Ad strain dl7001. Ad dl7001 is an Ad5 variantthat carries an approximately 3 kb deletion of the Ad5 sequence (GenBankAccession No. M73260) between m.u. 78.4 through 86. The CMV/rabiesglycoprotein/pA minicassette of pAd.CMVrab is inserted between deletedadenovirus m.u.1 and 9, with the remaining Ad5 m.u. 9-100 having theabove-mentioned E3 gene deletion. Restriction endonuclease enzymes arerepresented by conventional designations.

FIG. 2 is a bar graph plotting ³H-thymidine ([3H]TdR) incorporation,measured at counts per minute±standard deviation (cpm±SD), forirradiated splenocytes plated at 5×10⁵ cells per well of a round bottommicrotiter plate and incubated with 5 (diagonally striped), 1(cross-hatched) or 0.2 (solid) μg/ml of betapropionolactone-inactivatedEvelyn Rockitniki Abelseth rabies strain (ERA-BPL) or approximately 1(diagonally striped), 0.1 (cross-hatched), and 0.01 (solid) pfu ofAdrab.gp per cell or medium only as a negative control for 60 minutes at37° C. As described in Example 2B, after cloned T cells were added,cells were pulsed two days later for 6 hours with ³H-thymidine,harvested and counted in a β-counter.

FIG. 3A is a graph plotting % specific lysis (means of triplicates±SD)vs. effector:target cell ratio for groups of C3H/He mice inoculated with2×10⁶ pfu of Adrab.gp (solid box) or H5.020CMVlacZ (open box), asdescribed in Example 4B. Splenocytes were harvested 14 days later andco-cultured for 5 days with 1 pfu of Adrab.gp virus per cells. Activatedlymphocytes were then tested at different E:T ratios on H-2 compatibleL929 cells stably transfected with a rabies virus G protein-expressingvector (t.L929rab.gp) in a 4 hour ⁵¹Cr-release assay.

FIG. 3B is a graph of an experiment similar to FIG. 3A, but in which theactivated lymphocytes were tested at different E:T ratios on H-2compatible L929 cells stably transfected with a neomycin-expressingvector (t.L929.neo) in the ⁵¹Cr-release assay, as a control.

FIG. 4A is a graph plotting number of cells vs. intensity offluorescence for L929 fibroblasts plated in 24-well Costar plates inmedium supplemented with 2% 35 fetal bovine serum (FBS) followinginfection with 1 pfu/cell of VRG, as described in Example 5 below. Cellsharvested 12 hours after infection and stained by indirectimmunofluorescence with monoclonal antibody (MAb) 509-6 were analyzed byfluorescence activated cell sorting (FACS). The line on the graphlabeled “B” is the threshold below which 99% of the population arenegative. Line “C” represents the region that encompasses all events onthe histogram.

FIG. 4B is a graph similar to FIG. 4A above, except the cells wereharvested 36 hours after infection.

FIG. 4C is a graph similar to FIG. 4A above., except the cells wereharvested 60 hours after infection.

FIG. 4D is a graph similar to FIG. 4A above, except the cells, harvested12 hours after infection, were stained using cells treated only with thefluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin(Ig) as a control.

FIG. 4E is a graph similar to FIG. 4D above, except the cells wereharvested 36 hours after infection.

FIG. 4F is a graph similar to FIG. 4D above, except the cells wereharvested 60 hours after infection.

FIG. 4G is a graph similar to FIG. 4A above, except the cells wereinfected with 1 pfu Adrab.gp virus, and cells were harvested 12 hoursafter infection.

FIG. 4H is a graph similar to FIG. 4G, except the cells were harvested36 hours after infection.

FIG. 4I is a graph similar to FIG. 4G, except the cells were harvested60 hours after infection.

FIG. 4J is a graph similar to FIG. 4G above, except the cells werestained by indirect immunofluorescence using cells treated only withFITC-labeled goat anti-mouse Ig as a control.

FIG. 4K is a graph similar to FIG. 4J above, except the cells wereharvested 36 hours after infection.

FIG. 4L is a graph similar to FIG. 4J above, except the cells wereharvested 60 hours after infection.

FIG. 5A is a graph plotting optical density at 405 nm vs. serum dilutionfor duplicate samples±SD, as described in Example 6B below for miceimmunized with a replication-competent E3 deleted adenovirus (open box)or Adrab.gp (solid box). Native age-matched control mice were used ascontrols (X). Mice were bled 10 days after immunization and serumantibody titers to adenoviral antigens were determined by an ELISA onplates coated with 1 μg/mL of purified H5.020CMVlacZ virus.

FIG. 5B is a graph similar to that of FIG. 5A for mice immunized asdescribed in FIG. 6A below, and bled at 16 days.

FIG. 6A is a graph plotting mean percentage (%) specific lysis oftriplicates±SD vs. E:T cell ratio for C3H/He mice inoculated with 10⁶pfu of replication competent E3 deleted adenovirus and boosted 3 weekslater with Adrab.gp (open box). Control mice were inoculated withAdrab.gp only (solid box). Mice were sacrificed 4 weeks later and uponrestimulation with 1 pfu of Adrab.gp per cell tested on a 4 hour⁵¹Cr-release assay on L929 cells stably transfected with pSG5rab.gp. SeeExample 6.

FIG. 6B is a graph similar to FIG. 6A, except the L929 cells weretransfected with pSV2neo.

FIG. 7 is a graph plotting % survival of vaccinated mice vs. days afterchallenge with rabies virus. Mice were challenged 3 days (opentriangle), 7 days (open square), and 10 days (solid square) aftervaccination. X represents naive mice controls. See, Example 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods of effectivelyinducing a protective immune response to a disease agent. Thecompositions include a recombinant replication-defective adenovirus, andpharmaceutical and veterinary compositions containing the rAd. The rAdbackbone was previously used for gene therapy. As discussed herein, theinventors have surprisingly found that use of such a recombinant Ad,described in detail below, provides substantially complete immuneprotection in vaccinates.

By “substantially complete” protection is meant when administered in aneffective amount, the recombinant adenovirus presents an immunogenicprotein in such a manner that a protective immune response is observedin substantially all vaccinates after a single administration. By“substantially all” is meant greater than 90% of the vaccinates.Unexpectedly, the recombinant vaccine permits successful vaccinationwith very few booster administrations. Also unexpectedly, therecombinant vaccine permits vaccination at an unexpectedly lower dosagethan is normally used in similar vaccines in which the same protein ispresent in another recombinant virus. For example, immunization of micewith a single dose of as little as 10⁴ pfu of the recombinant,replication defective Ad containing a rabies glycoprotein has beenobserved to induce complete protection against rabies infection. Partialprotection was seen seven days after immunization.

While not wishing to be bound by theory, the inventors currently believethat this recombinant, replication defective Ad vaccine is advantageousover, e.g. , the vaccinia vaccine, because it permits lower doses ofantigen to be expressed for an extended period of time by a non-lyticvirus. For example, although vaccinia expresses higher doses of antigen,e.g., a rabies antigen, it is a lytic virus which causes a rapid demiseof infected cells. The finding that the recombinantreplication-defective Ad, e.g., Adrab.gp virus, used in the method ofthe present invention is more efficacious than the currently usedvaccinia rabies (VRG) vaccine is unexpected and incompatible withcurrent thinking that the antigenic dose governs the magnitude of theimmune response. The use of the recombinant replication defectiveadenovirus also confers safety and efficacy advantages over othervaccine carriers, such as vaccinia. The adenovirus construct results inslow accumulation of the rabies virus G protein on the surface ofinfected cells without causing visible cell damage (data not shown). Incontrast, cells infected with VRG recombinant rapidly expresssubstantial amounts of the rabies virus G protein on the cell surfacebut then die shortly after infection. The adenoviral construct persistsfor at least seven days in immunocompetent mice.

With respect to safety, the present invention provides a recombinantreplication-defective Ad which is thus highly unlikely to spread withina host or among individuals, particularly in view of the fact that therecombinant, E1-deleted dl7001 Ad virus, which is the backbone of theexemplary replication defective recombinant Ad used in the examplesbelow has already been approved for use in humans for gene therapy,i.e., for the replacement of faulty or missing genes. The recombinantvirus lacks oncogenic potential because the E1 gene that can function asan oncogene in some adenovirus strains has been deleted. Further, cellsinfected with the recombinant, replication defective adenovirus arecompletely eliminated by CD8 T cells within 21 days in immunocompetenthosts.

With respect to efficacy, the recombinant, replication defective Ad ofthis invention is highly efficacious at inducing cytolytic T cells andantibodies to the inserted heterologous protein expressed by the virus.This has been demonstrated with a recombinant, replication defective Adcontaining a sequence encoding the rabies virus glycoprotein as theheterologous gene, which Ad has been administered to animals by otherthan the oral route.

The recombinant virus of this invention is also surprisingly moreeffective as a vaccine than other, previously reported, replicationdefective adenovirus vaccines. See, for example, Ragot et al, Eliot etal, and Jacobs et al, all cited above. In contrast to the otherreplication defective adenovirus vaccines, the vaccine compositionuseful in the present invention can be used at lower doses. This vaccinecan also be administered in a single inoculation to obtain substantiallycomplete protection.

For these reasons, the recombinant replication-defective adenovirus ofthe invention and particularly the preferred embodiment which makes useof the pAdCMV.lacZ (or H5.020CMVlacZ) Ad vector described below, can beused as a prophylactic or therapeutic vaccine against any pathogen forwhich the antigen(s) crucial for induction of an immune response able tolimit the spread of the pathogen has been identified and for which thecDNA is available.

I. The Recombinant Adenovirus

As used herein, the term “minicassette” refers to the nucleotidesequence comprised of (a) a non-Ad promoter, which directs thereplication and expression of (b) the following nucleotide sequencewhich encodes a heterologous protein immunogen, which is followed by (c)a polyA nucleotide sequence. By “vector or plasmid” is meant theconstruct comprised of 5′ sequences of the Ad virus (usually Ad m.u.0-1) deleted of the E1 gene (which occurs between Ad m.u. 1-9), whichmay contain a heterologous nucleotide sequence, but which does notcontain the 3′ end of the Ad virus (generally between about Ad m.u. 16to 100), but rather conventional plasmid sequences. This vector does notcontain all of the genes essential to a replicative virus. By“recombinant, replication defective Ad” is meant the infectiousrecombinant virus, deleted of its E1 gene, into which location isinserted the minicassette, and which contains all of the 3′ sequencesessential to an infectious virus except for a functional deletion in theE3 gene region.

The recombinant virus of the method of the invention is areplication-defective recombinant adenovirus containing a deletion ofits E1 gene and at least a partial, functional deletion of its E3 gene.In the site of the E1 deletion a minicassette is inserted, whichcomprises a nucleotide sequence encoding a heterologous proteinimmunogen and a non-adenovirus promoter directing the replication andexpression of the nucleotide sequence encoding the heterologous protein.

Any Ad that infects the target cells is appropriate for use in thisinvention. Desirable adenoviruses are human type C adenoviruses,including serotypes Ad2 and Ad5. The DNA sequences of a number ofadenovirus types, including type Ad5, are available from GenBank[Accession No. M73260]. The adenovirus sequences may be obtained fromany known adenovirus type, including the presently identified 41 humantypes [Horwitz et al, Virology, 2d ed., B. N. Fields, Raven Press, Ltd.,New York (1990)]. Similarly, adenoviruses known to infect other animalsmay also be employed in this invention. The selection of the adenovirustype and strain is not anticipated to limit the following invention. Avariety of adenovirus strains are available from the American TypeCulture Collection, Rockville, Md., or available by request from avariety of commercial and institutional sources. In the followingexemplary embodiment, an adenovirus type 5 (AdS) sequence obtained fromGenBank [Acc. No. M73260] is used for convenience.

Adenoviruses of the present invention are replication defective, i.e.,intact adenoviruses which have been rendered replication defective bydeleting the early gene locus that encodes E1a and E1b. See, K. F.Kozarsky and J. M. Wilson, Curr. Opin. Genet. Dev., 3:499-503 (1993).Similarly, a replication defective adenovirus may be designed bydeleting less than the entire E1a and E1b locus, but enough tofunctionally disable the E1genes.

An additional characteristic of the Ad useful in this invention is thatthe E3 gene is deleted, i.e., from about m.u. 78.5 to about m.u. 84.3 ofAd5. While the presently preferred embodiment contains a completedeletion of that sequence, it may be possible to partially delete the E3sequence to disable the functional abilities of the E3 gene.

A preferred recombinant Ad virus may be produced by using a plasmidvector pAd.CMVlacZ as described in FIG. 1B. This plasmid containsadenovirus sequences Ad m.u. 0-1 (i.e., it is fully deleted of E1a andE1b genes), after which a selected minigene may be inserted, e.g., therabies glycoprotein under control of a heterologous promoter and otherregulatory sequences, if desired, followed by the sequence Ad m.u.9 to16 and plasmid sequences. When this vector is manipulated to place aminicassette into the E1 deletion site, and supplied with the remaining3′ Ad sequences with a full deletion of E3 and cultured in a helper cellline, the resulting recombinant adenovirus is capable of functioning asa rabies vaccine. This recombinant virus, called Adrab.gp orH5020.CMVrab, is described in detail in Example 1 and in flow chart formin FIGS. 1A through 1D.

The preferred recombinant Ad of this invention contains a minicassettewhich uses the cytomegalovirus (CMV) promoter [see, e.g., Boshart et al,Cell, 41: 521-530 (1985)] to control the expression of the insertedheterologous gene. The promoter is inserted in the site of the E1deletion and directs the replication and expression of the proteinencoded by the selected heterologous gene. However, this invention isnot limited by the selection of the promoter, except that the promotershould be heterologous to the Ad virus, i.e., the E1 Ad promoter isreplaced using techniques known to those of skill in the art. Otherdesirable promoters include the Rous sarcoma virus LTRpromoter/enhancer, the SV40 promoter, and the chicken cytoplasmicβ-actin promoter [T. A. Kost et al, Nucl. Acids Res., 11(23):8287(1983)]. Still other promoter/enhancer sequences may be readily selectedby one of skill in the art.

As discussed above, in the site of the E1 deletion, and under control ofa promoter heterologous to Ad, a nucleic acid sequence, preferably inthe form of DNA, encoding a protein heterologous to the Ad is insertedusing techniques known to those of skill in the art.

The heterologous nucleic acid encodes a protein which is desirablycapable of inducing an immune response to a pathogen. Such a protein maybe a protein from rabies virus, human papilloma virus, humanimmunodeficiency virus (HIV), respiratory syncytial virus (RSV). Thevaccine method of the present invention may also be employed with atumor-associated protein specific for a selected malignancy. These tumorantigens include viral oncogenes, such as E6 and E7 of human papillomavirus or cellular oncogenes such as mutated ras or p53. Particularly,where the condition is human immunodeficiency virus (HIV) infection, theprotein is preferably HIV glycoprotein 120 for which sequences areavailable from GenBank. Where the condition is human papilloma virusinfection, the protein is selected from the group consisting of E6, E7and/or L1 [Seedorf, K. et al, Virol., 145:181-185 (1985)]. Where thecondition is respiratory syncytial virus infection, the protein isselected from the group consisting of the glyco- (G) protein and thefusion (F) protein, for which sequences are available from GenBank. Inaddition to these proteins, other virus-associated proteins are readilyavailable to those of skill in the art. Selection of the heterologousproteins is not a limiting factor in this invention.

In a particularly preferred embodiment, the condition is rabies and theprotein is the rabies glycoprotein [see, U.S. Pat. No. 4,393,201]. Avariety of rabies strains are well known and available from academic andcommercial sources, including depositaries such as the American TypeCulture Collection, or may be isolated using known techniques. Thestrain used in the examples below is the Evelyn Rockitniki Abelseth(ERA) strain. However, this invention is not limited by the selection ofthe rabies strain.

In a preferred embodiment, cDNA encoding the rabies virus glycoproteinis inserted under control of a CMV promoter into the pAdCMV.lacZ (orH5.020CMVlacZ) Ad vector and supplied with the essential genes forinfectivity and viral formation in a helper cell line using standardtechniques, as described in detail in Example 1. Immunization studiesrevealed that a single administration of the resulting recombinantreplication defective virus conferred complete protection at arelatively low dose following challenge with rabies virus.

II. Formulation of Vaccine

A recombinant replication defective Ad bearing a gene encoding animmunogenic protein may be administered to a human or veterinarypatient, preferably suspended in a biologically compatible solution orpharmaceutically acceptable delivery vehicle. A suitable vehicle issterile saline. Other aqueous and non-aqueous isotonic sterile injectionsolutions and aqueous and non-aqueous sterile suspensions known to bepharmaceutically acceptable carriers and well known to those of skill inthe art may be employed for this purpose.

Optionally, a vaccinal composition of the invention may be formulated tocontain other components, including, e.g. adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art.

III. Administration of Vaccine

The recombinant, replication defective viruses are administered in an“effective amount”, that is, an amount of recombinant virus that iseffective in a route of administration to transfect the desired cellsand provide sufficient levels of expression of the selected gene toprovide a vaccinal benefit, i.e., protective immunity.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, rectal, oral and other parental routes of administration.Routes of administration may be combined, if desired, or adjusteddepending upon the immunogen or the disease. For example, in prophylaxisof rabies, the subcutaneous, intratracheal and intranasal routes arepreferred. The route of administration primarily will depend on thenature of the disease being treated.

Doses or effective amounts of the recombinant replication defective Advirus will depend primarily on factors such as the condition, theselected gene, the age, weight and health of the animal, and may thusvary among animals. For example, a prophylactically effective amount ordose of the Ad vaccine is generally in the range of from about 100 μl toabout 10 ml of saline solution containing concentrations of from about1×10⁴ to 1×10⁷ plaque forming units (pfu) virus/ml. A preferred dose isfrom about 1 to about 10 ml saline solution at the above concentrations.The levels of immunity of the selected gene can be monitored todetermine the need, if any, for boosters.

Currently, when vaccinating against rabies, the preferred dose is about10⁴ pfu of the recombinant virus per mouse, preferably suspended inabout 0.1 mL saline. Thus, when vaccinating against rabies infection, alarger animal would preferably be administered about a 1 mL dosecontaining about 1×10⁵ Adrab.gp pfu suspended in saline. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

The following examples illustrate the preferred methods for preparingthe vectors and the recombinant viruses used in the vaccine and methodof the invention. These examples are illustrative only and do not limitthe scope of the invention.

Example 1 —Production and Purification of Vectors and Viruses

A. Adrab.gp

A recombinant, replication defective adenovirus expressing the rabiesvirus G protein of the Evelyn Rockitniki Abelseth (ERA) strain of rabiesvirus [ATCC VR-332; U.S. Pat. No. 3,423,505] (ERA) was constructed asfollows. See the flowchart of FIGS. 1A to 1D.

The 1650 bp rabies virus G cDNA (nucleotides 1178 to 2827 of SEQ IDNO: 1) was purified from the pSG5rab.gp plasmid [S. R. Burger et al, J.Gen. Virol., 72:359-367 (1991)] upon digestion with BglII, andblunt-ended with Klenow to supply the G gene. See also U.S. Pat. No.4,393,201, issued Jul. 12, 1983.

The pAd.CMVlacZ vector [J. Wilson et al, Hum. Gene Ther., 5:501-519(1994); K. Kozarsky et al, J. Biol. Chem., 269:13695-13702 (1994)],which contains Ad5 m.u. 0-1, followed by the cytomegalovirus (CMV)enhancer/promoter, the beta galactosidase (lacz) gene, a polyadenylationsignal (pA), adenovirus m.u. 9-16 and plasmid sequences from plasmidpAT153 including an origin of replication and ampicillin resistancegene, was completely digested with NotI to remove the lacZ gene andprovide an ^(˜)5.6 kb backbone.

The cDNA encoding the rabies G protein, described above, was insertedinto this 5.6 kb fragment via blunt-end cloning to generatepAdCMV.rabgp, which is similar to pAd.CMVlacZ but contains the rabiessequence in place of the lacZ gene. The appropriate orientation of theinsert was confirmed by restriction enzyme mapping. pAdCMV.rabgp [SEQ IDNO: 1] contains adenovirus m.u. 0-1 (nucleotides 12 to 364 of SEQ ID NO:1); followed by a cytomegalovirus enhancer/promoter (nucleotides 382 to863 of SEQ ID NO: 1); the rabies glycoprotein gene (nucleotides 1178 to2827 of SEQ ID NO: 1); a polyadenylation signal (nucleotides 2836-3034of SEQ ID NO: 1) ; adenovirus m.u. 9-16 (nucleotides 3061 to 5524 of SEQID NO: 1); and plasmid sequences from plasmid pAT153 (nucleotides 5525to 8236 of SEQ ID NO: 1). The remaining nucleotides of SEQ ID NO: 1 arethe result of cloning and plasmid construction.

To provide a recombinant virus capable of infecting a cell, the 3′ endof the adenovirus sequence was needed to replace the pAT153 plasmidsequences of pAdCMV.rabgp. The plasmid pAdCMV.rabgp was linearized withNheI. The linearized plasmid was co-transfected into 293 packaging cells[ATCC CRL 1573] which contain and express the transforming genes ofhuman adenovirus type 5 to allow replication of the adenovirus [F. L.Graham et al, J. Gen. Virol., 36:59-72 (1977)]. The transfectedpackaging cells were grown in DMEM with 10% FBS without HEPES buffer ina 5% CO₂ incubator with an E3 deleted Ad5 DNA [Ad5dl7001, a variant thatcarries a 3 kb deletion between m.u. 78.4 through 86 in the nonessentialE3 region (provided by Dr. William Wold, Washington, University, St.Louis, Mo.)]. This Ad5dl7001 had been digested with a restriction enzymeClaI to remove the left end, i.e., 917 bp from the 5′ end of theadenovirus sequence, rendering the DNA non-infectious.

Following the co-transfection, only products of homologous recombinationwhich occurred between Ad m.u. 9-16 of the pAdCMV.rabgp and the 5′deleted-Ad5dl7001 could produce replicative Ad virus in 293 cells. Thatis, when homologous recombination occurred, the 3′ end of pAd.rabgp fromabout Ad m.u. 9 to about m.u. 16 and all of the plasmid sequence wasswapped with the 3′ end of the 5′ truncated Ad5dl7001 virus, from aboutAd m.u. 9 through m.u. 100.

Several recombinant viral plaques were harvested and tested forexpression of the rabies virus G protein as described below. Onerecombinant, replication defective clone termed Adrab.gp was purified bytwo rounds of plaque purification and used for further studies and isillustrated schematically in FIG. 1D above.

The recombinant, replication defective Ad Adrab.gp contains Ad5 m.u.0-1, followed by the CMV enhancer/promoter, the rabies G gene, a pAsite, and Ad5 m.u. 9-78.4 and 86-100.

B. H5.010 CMVlacZ

The recombinant replication defective Ad, H5.010CMVlacZ, issubstantially identical to Adrab.gp, except that this virus contains E.coli lacZ in place of the rabies G protein and only a partial deletionof E3.

The plasmid pAd.CMVlacZ described above, was linearized with NheI andco-transfected into 293 cells with a partially E3 deleted Ads DNA (sub360 DNA, H5sub360), which had been digested with ClaI to eliminate thesequence of m.u. 83.5 to 85. As above, homologous recombination,followed by plaquing and harvesting produced the resulting recombinantadenovirus, designated H5.010CMVlacZ. This virus contains the sequencefrom Ad5 m.u. 0-1, followed by the CMV enhancer/promoter, theEscherichia coli lacz gene, a pA site, and Ad5 m.u. 9-83.5 and 85-100.

C. Viral Propagation and Purification

The adenoviral recombinants, Adrab.gp H5.010CMVlacZ, and Ad5dl7001, areplication competent adenovirus, on 293 cells for 72 hours. Virus wasrecovered on the third round of freeze-thawing. Cell-free supernatantswere either used directly or they were further purified by CsCl densitycentrifugation. Viral stocks were titrated on 293 cells using a plaqueassay.

Example 2—Immunofluorescence and T Cell Studies

To confirm that the Adrab.gp recombinant virus expresses the rabiesvirus G protein on infected cells in a form recognized by antibodies andcytolytic T cells directed against rabies virus, a series of in vitroexperiments were performed initially.

A. Indirect Immunofluorescence

To assess the conformation of the G protein as expressed by the Adrab.gpvirus, HeLa cells [which had been maintained in Dulbecco's minimalessential medium (DMEM) supplemented with 10% FBS, HEPES buffer andantibiotics in a 10% CO₂ incubator] were infected for 48 hours with 1pfu of Adrab.gp virus per cell or as a control with H5.020CMVlacZ. Cellswere stained 24 hours later by an indirect immunofluorescence assayusing three MAbs (designated 523-11, 509-6, and 1112-1, and preparedusing a 1:100 to 1:1000 dilution of ascitic fluid) to differentconformation-dependent binding sites of the rabies virus G protein. TheB cell hybridoma cells 509-6, 1112-1, and 523-11 secrete antibodies todifferent antigenic sites of the rabies virus G protein (509-6 to siteI, 1112-1 to site II, and 523-11 to site III [T. J. Wiktor et al, Proc.Natl. Acad. Sci. USA, 75:3938-3945 (1978)]. These hybridoma cells weregrown in DMEM supplemented with 10% FBS. Ascetic fluid was prepared inBALB/c mice. The assay was performed as follows.

The HeLa cells were infected for various times with 1 pfu of recombinantadenovirus or with 1 pfu of the vaccinia VRG virus described above percell in 24-well Costar plates seeded with 5×10⁵ cells per well. Cellswere harvested at varied times after infection by treatment with trypsinand incubated for 60 minutes on ice with the MAbs identified above.Cells were washed once with phosphate-buffered saline (PBS) and thenincubated with a FITC-labeled goat anti-mouse immunoglobulin (Ig)antibody. Cells were washed and analyzed by a fluorescence activatedcell sorter (FACS). Alternatively cells adherent to glass cover slipswere stained with the same antibody preparations for subsequent analysiswith confocal microscopy.

For all of the antibodies, Adrab.gp virus-infected cells exhibitedsurface staining with the antibody, while cells infected with thecontrol recombinant virus expressing lacZ were negative.

B. T Cell Proliferation Assay

Further in vitro studies showed that the recombinant virus Adrab.gpinduced proliferation of a rabies virus G protein specific T helper cellclone in the presence of syngeneic, γ-irradiated splenocytes (FIG. 2).In a separate experiment, this T cell clone did not proliferate in thepresence of H5.010CMVlacZ (data not shown).

A rabies virus-specific helper T cell clone, obtained from splenocytesof VRG immune C3H/He mice in the inventors laboratory, was cultured(2×10⁴ cells/well) in 96-well round-bottom microtiter plate with 5×10⁵irradiated syngeneic C3H/He splenocytes pretreated with differentantigen preparations (1, 0.1 and 0.01 pfu Adrab.gp per cell) in DMEMsupplemented with 2% PBS and 10⁻⁶ M 2-mercaptoethanol and 10% ratConcanavalin A supernatant as a lymphokine source as describedpreviously [L. Otvos, Jr., Biochim. Biophys. Acta, 1224:68-76 (1994)].Proliferation of the cloned T cells was assessed 48 hours later by a 6hour pulse with 0.5 μCi of ³H-thymidine as described in H. C. J. Ertl etal, Eur. J. Immunol., 21:1-10 (1991). Furthermore, mouse fibroblastsinfected with the Adrab.gp recombinant virus were rendered susceptibleto lysis by rabies virus G protein induced H-2 compatible cytolytic Tcells.

Together these in vitro experiments demonstrated that Adrab.gp causesexpression of the rabies virus G protein in a form that is readilyrecognized by both rabies virus-specific antibodies and T cells of thehelper and the cytolytic subset. Specificly, FIG. 2 illustrates thatAdrab.gp induces proliferation of a rabies virus G protein T helper cellclone in the presence of antigen presenting cells.

Example 3—Immunization Studies In the next set of experiments, mice wereimmunized with the Adrab.gp recombinant virus at several doses usingdifferent routes of immunization as follows.

Groups of eight to twelve week old outbred ICR [Harlan Sprague-Dawley(Indianapolis, In.)] or C3H/He mice [The Jackson Laboratories (BarHarbor, Me.)] were injected subcutaneously (s.c.), orally (per os),intranasally (i.n.), or upon anesthesia and surgical exposure of thetrachea intratracheally (i.t.), with the recombinant adenoviruses of theprevious examples diluted in 100 to 150 μl of saline. VRG [which hadbeen propagated on HeLa cells as described in T. J. Wiktor et al, Proc.Natl. Acad. Sci. USA, 81:7194-7198 (1984)] was given s.c. Mice were bledby retro-orbital puncture in regular intervals after immunization toassess serum antibody titers.

The challenge virus standard (CVS)-24 strain of rabies virus, that isantigenically closely related to the ERA strain but shows highervirulence in mice, was derived from brain suspensions of infectednewborn ICR mice [T. J. Wiktor et al, J. Virol., 21:626-633 (1977]. Micewere challenged with 10 mean lethal doses (LD₅₀) of CVS-24 virus givenintramuscularly (i.m.) into the masseter muscle; they were observed forthe following 3 weeks for symptoms indicative of a rabies virusinfection. Mice that developed complete bilateral hind leg paralysis(proceeding death by 24 to 48 hours) were euthanized for humanitarianreasons.

A. Virus Neutralizing Antibodies

Groups of ICR mice were immunized in three separate experiments with thedifferent recombinant viruses given at the doses in Table 1 below eitheri.m., i.n., i.t., or per os. Mice inoculated into the trachea or i.n.were anesthetized prior to vaccination. Mice were bled 10 to 14 dayslater after a single immunization and serum antibody titers to rabiesvirus were tested by a neutralization assay. Virus neutralizing antibody(VNA) titers were determined on BHK-21 cells using infectious ERA virusat 1 pfu per cell [B. D. Dietzschold et al, Virology, 161:29-36 (1987)].

Table 1 below illustrates the data expressed as neutralization titerswhich are the reciprocal of the serum dilution resulting in a 50%reduction in the number of infected cells. Samples were assayed induplicate in serial 3-fold dilutions starting with a dilution of 1:5.Standard deviations were within 10% for any given experiment.

As illustrated by the results in Table 1, virus given s.c., i.t., ori.n. induced a potent neutralizing antibody response if given at 10⁶pfu. Oral immunization with Adrab.gp or systemic immunization withH5.020CMVlacZ failed to induce a measurable antibody response to rabiesvirus. The antibody responses to different doses of the recombinantreplication-defective Adrab.gp were clearly superior to the responseinduced by the VRG recombinant. For example, the antibody titers of miceinoculated with as little as 2×10⁴ pfu of Adrab.gp were more than 10times higher than those of mice infected with 2×10⁶ pfu of VRG (Table1).

TABLE 1 Adrab.gp Recombinant Induces Neutralizing Antibodies to RabiesVirus Route of Time VNA titer Vaccine Dose Immunizat'n After Immunizat'nAdrab.gp 2 × 10⁶ s.c. day 10 3,645 Adrab.gp 2 × 10⁵ s.c. day 10 405Adrab.gp 2 × 10⁴ s.c. day 10 405 VRG 2 × 10⁶ s.c. day 10 45 VRG 2 × 10⁵s.c. day 10 15 VRG 2 × 10⁴ s.c. day 10 5 None — — day 10 <5 Adrab.gp 10⁴s.c. day 14 1,215 Adrab.gp 10³ s.c. day 14 405 Adrab.gp 10² s.c. day 14<5 Adrab.gp 10⁶ i.n. day 14 1,215 Adrab.gp 10⁶ i.t. day 14 3,645Adrab.gp 10⁶ per os day 14 <5 None — — <5

To ensure that the antibody response was caused by infection recombinantvirus rather than by G protein fragments contaminating thevirus-containing tissue culture supernatant used for immunization, micewere vaccinated with an equal dose of PFUs of unpurified and gradientpurified recombinant adenovirus. Both groups of mice developed identicalvirus neutralizing antibody titers.

B. Cell-mediated Cytolysis

In addition to neutralizing antibodies, mice inoculated s.c. withAdrab.gp virus developed rabies virus G protein-specific cytolytic Tcells able to kill H-2 compatible L929 target cells stably transfectedwith a plasmid vector expressing the rabies virus G protein under thecontrol of the SV40 early promoter [Z. Q. Xiang et al, J. Virol. Meth.,47:103-116 (1994)].

L929 mouse fibroblasts were maintained in Dulbecco's minimal essentialmedium (DMEM) supplemented with 10% fetal bovine serum (FBS), HEPESbuffer and antibiotics in a 10% CO₂ incubator. L929 cells stablytransfected with pSG5rab.gp [S. R. Burger et al, cited above],expressing the rabies virus G protein as well as L929 cells transfectedwith pSV2neo [ATCC Accession No. 37149] were maintained in 10% DMEMsupplemented with 10% FBS. These cell lines used as target cells forcell-mediated cytolysis assays have been described in detail previously[Z. Q. Xiang et al, J. Virol. Meth., 47:103-116 (1994)].

Briefly, splenocytes were harvested from immunized C3H/He mice. Singlecells were prepared and incubated at 6×10⁶ cells per well with 1 pfu percell of the Adrab.gp recombinant virus in 1.6 ml of DMEM supplementedwith 10⁻⁶ M 2-mercaptoethanol and 2% FBS for 5 days in a humidified 10%CO₂ incubator. The effector cells were then co-cultured with⁵¹Cr-labeled L929 cells expressing the rabies virus G protein uponstable transfection with the pSG5rab.gp vector at variedeffector-to-target cells ratios. To assess spontaneous release,⁵¹Cr-labeled target cells were incubated with medium; to determinemaximal release target cells were co-cultured with 10% sodium dodecylsulfate. Cell-free supernatants were harvested 4 hours later andradioactivity was measured. Percentage of specific lysis was calculatedby using the formula [Y. Yang et al, Immunity, 1:433-442 (1994)]:100 × [(Release  in  presence  of  effectors − spontaneous  release)/(Maximal  release − spontaneous  release)]

The results are illustrated graphically in FIG. 3A, which illustratesthat the Adrab.gp construct induces cytolytic T cells to the rabiesvirus G protein. See, also the results of FIG. 3B, in which lymphocyteswere tested at different E:T ratios on an L929 cell line transfectedwith Adrab.gp or a neomycin expressing control.

Example 4—Challenge Studies

Four different experiments were conducted in which mice, immunized asdescribed in Example 3A above, were challenged with 10 LD₅₀ of rabiesvirus. Briefly, mice immunized with the Adrab.gp or the VRG recombinantvirus were challenged 2 to 5 weeks after immunization with 10 LD₅₀ ofthe virulent CVS-24 strain of rabies virus given i.m. into the massetermuscle. Mice that subsequently developed complete bilateral hind legparalysis indicative of a terminal rabies virus infection wereeuthanized for humanitarian reasons. Survivors were observed for a totalof 21 days.

The results are illustrated in Table 2 below. Mice immunized withAdrab.gp i.m., i.t., or i.n. using doses ranging from 10⁴ to 2×10⁶ pfuwere fully protected against infection; 87% of mice inoculated with 10³pfu were protected. All mice immunized with only 10² pfu of therecombinant adenovirus or inoculated with the H5.020CMVlacZ controlvirus (2×10⁶ pfu) or with Adrab.gp per os developed a fatal rabies virusencephalitis within 10 days after infection. Mice vaccinated with VRGshowed partial protection; the group receiving the highest dose, i.e.,2×10⁶ pfu, had a mortality rate above 50% raising to ˜90% in miceinoculated with 2×10⁴ pfu of VRG.

TABLE 2 Adrab.gp Recombinant Virus Induces Protective Immunity toChallenge with Rabies Virus Route of % Vaccine Dose immunizationmortality Adrab.gp 2 × 10⁶ s.c. 0 H5.010CMVlacZ 2 × 10⁶ s.c. 90 Adrab.gp2 × 10⁶ s.c. 0 Adrab.gp 2 × 10⁵ s.c. 0 Adrab.gp 2 × 10⁴ s.c. 0 VRG 2 ×10⁶ s.c. 56 VRG 2 × 10⁵ s.c. 71 VRG 2 × 10⁴ s.c. 86 None — — 100Adrab.gp 10⁴ s.c. 0 Adrab.gp 10³ s.c. 13 Adrab.gp 10² s.c. 100 None — —100 Adrab.gp 10⁶ i.n. 0 Adrab.gp 10⁶ i.t. 0 Adrab.gp 10⁶ per os 100 None— — 100

Example 5—Comparison Studies

The relationship between the magnitude of an immune response and theamount of antigen available to induce naive T and B cells was studied.As determined by immunofluorescence and subsequent analysis by FACS(FIGS. 4A-4L), both the VRG and the Adrab.gp recombinants expresscomparable levels of the rabies virus G protein but the kinetics ofexpression are different. Cells infected with the VRG virus express highlevels of G protein within 12 hours after infection; these levelsincreased over the next day. By 60 hours the VRG virus has completelylysed a cell population infected with ˜1 pfu of virus per cell.

The same cell line infected with 1 pfu of Adrab.gp per cell shows lowexpression of the rabies virus G protein on day 1. The level ofexpression increases until days 3 to 4 after infection and then reachesplateau levels (data shown for days 1 to 3 in FIG. 4A through FIG. 4L).The replication-defective recombinant adenoviruses are non-lytic andmaintain stable infection and expression of virus-encoded proteins forextended periods of time. In tissue culture, expression has been shownfor 7 days in vivo; using the H5.010CMVlacZ recombinant virus, stablelevels of expression were demonstrated in immunocompromised mice for 10months.

A non-lytic virus, e.g., the recombinant replication defectiveadenovirus, that expresses antigens for prolonged periods of time mightthus be more immunogenic compared to a replicating agent that causesdeath of the infected cells within 24 to 48 hours, e.g., vaccinia.

To substantiate this hypothesis, the inventors compared the immuneresponse to rabies proteins upon immunization of mice with areplication-defective E1 deleted adenovirus and a replication-competentadenovirus. Both adenoviruses were of the human strain 5 and both weredeleted in E3. These recombinant viruses were tested by enzyme linkedimmunoadsorbent assay (ELISA) (FIGS. 5A and 5B). The ELISAs wereconducted in 96-well microtiter plates coated with 0.1 to 0.2 μg perwell of ERA-BPL virus or 1-2 pg per well of purified H5.010CMVlacZvirus, using an alkaline phosphatase conjugated goat anti-mouse Ig assecond antibody as described in detail in Xiang and Ertl, Virus Res.,24:297-314 (1992). As shown in FIGS. 5A and 5B, the antibody response tothe E1 deleted Adrab.gp virus (solid box) was superior to that of areplication competent Ad virus (open box). This supports the positionthat long-term expression of viral antigens by a non-lytic virus caninduce stronger immune response compared to short-term expression by areplication-competent agent. FIGS. 5A and 5B illustrate that expressionof E1 causes a reduction in the antibody response to adenovirus.

These studies demonstrate that the recombinant replication-defectiveadenovirus used in the present invention shows higher immunogenicitycompared to a replication-competent adenovirus. Without wishing to bebound by theory, it is believed that the length of expression of theantigen plays a role in induction of the immune response. In similarstudies comparing the replication defective adenovirus vaccine to theVRG vaccine, the Ad vaccine expresses the rabies antigen longer than theVRG recombinant virus vaccine.

Example 6—Further Comparative Studies

The following study was performed to test if pre-existing immunity toadenoviral proteins interferes with stimulation of a rabies Gprotein-specific immune response to the Adrab.gp construct. Groups ofC3H/He mice were immunized with 10⁵ or 10⁶ pfu of areplication-competent adenovirus human serotype 5 that had been deletedof the E3 gene. Mice were injected 4 weeks later with 10⁶ pfu ofAdrab.gp. Control mice were only injected with Adrab.gp (10⁶ pfu). Micewere bled 12 days later and neutralizing antibody titers were determined(Table 3).

TABLE 3 The Effect of Pre-Existing Immunity to Adenovirus on the RabiesVNA Response to the Adrab.gp Vaccine Pre-immunization Titer ImmunizationVNA None 10⁶ pfu Adrab.gp 3.645 10⁵ pfu Ad5d17001 10⁶ pfu Adrab.gp 3.64510⁶ pfu Ad517001 10⁶ pfu Adrab.gp 1.215 None None <5

Mice pre-immunized with adenovirus developed VNA to rabies virus uponbooster immunization with the Adrab.gp construct. Titers wereequivalent, or marginally lower, when compared to those in control micethat had only received Adrab.gp, indicating that antibodies toadenoviruses only marginally inhibit the B cell response to proteinsexpressed by adenovirus recombinants. When tested in comparison to areference serum provided by the World Health Organization, sera frompre-immune (both doses of adenovirus) or naive mice were shown to havetiters of 40 IU to rabies virus. Protection to rabies virus iscorrelated to antibody titers and 2 IU are considered sufficient toprotect against a severe challenge. Pre-immunity to adenovirus does,thus, not impair the ability of the Adrab.gp vaccine to elicitprotective immunity.

Similar data were obtained for the stimulation of cytolytic T cells torabies virus-infected cells, pre-immune animals showed somewhat lowerlysis compared to the control group (see FIGS. 6A and 6B). FIGS. 6A and6B illustrate that the cytolytic T cell response to rabies virus Gprotein expressing target cells upon immunization with Adrab.gp is onlyslightly reduced in animals immune to adenovirus. Nevertheless,adenovirus-immune mice still developed significant T cell responses tothe rabies virus G protein upon immunization with Adrab.gp.

Example 7—Additional Challenge Studies

In this experiment the kinetic of the induction of protective immunityupon vaccination was tested with the Adrab.gp virus. Vaccination torabies virus is in general given post-exposure, hence it is crucial forthe vaccine to induce a rapid immune response before the rabies virushas reached the central nervous system.

Mice were immunized with 106 PFU of Adrab.gp s.c. Immunized mice werechallenged 3 (⋄), 7 (□), and 10 (▪) days after vaccination with 10 LD₅₀of rabies virus given i.m. Naive mice (X) served as controls. Mice wereobserved for four weeks to record mortality. As shown in FIG. 7, micevaccinated with Adrab.gp virus 10 days previously were completelyprotected; while more than half of the animals were protected as earlyas seven days after a single injection. Mice vaccinated three daysbefore challenge succumbed to the infection.

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the compositions and processes of the present invention are believedto be encompassed in the scope of the claims appended hereto.

2 8236 base pairs nucleic acid double Not Relevant cDNA not provided CDS1185..2756 1 GAATTCGCTA GCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT 50ATGATAATGA GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG 100 GGCGGGTGACGTAGTAGTGT GGCGGAAGTG TGATGTTGCA AGTGTGGCGG 150 AACACATGTA AGCGACGGATGTGGCAAAAG TGACGTTTTT GGTGTGCGCC 200 GGTGTACACA GGAAGTGACA ATTTTCGCGCGGTTTTAGGC GGATGTTGTA 250 GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCGCGGGAAAACT 300 GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA350 ATATTTGTCT AGGGAGATCA GCCTGCAGGT CGTTACATAA CTTACGGTAA 400ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA 450 ATGACGTATGTTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 500 ATGGGTGGAG TATTTACGGTAAACTGCCCA CTTGGCAGTA CATCAAGTGT 550 ATCATATGCC AAGTACGCCC CCTATTGACGTCAATGACGG TAAATGGCCC 600 GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTCCTACTTGGCA 650 GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC700 AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT 750CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 800 GGACTTTCCAAAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG 850 GTAGGCGTGT ACGGTGGGAGGTCTATATAA GCAGAGCTCG TTTAGTGAAC 900 CGTCAGATCG CCTGGAGACG CCATCCACGCTGTTTTGACC TCCATAGAAG 950 ACACCGGGAC CGATCCAGCC TCCGGACTCT AGAGGATCCGGTACTCGAGG 1000 AACTGAAAAA CCAGAAAGTT AACTGGTAAG TTTAGTCTTT TTGTCTTTTA1050 TTTCAGGTCC CGGATCCGGT GGTGGTGCAA ATCAAAGAAC TGCTCCTCAG 1100TGGATGTTGC CTTTACTTCT AGGCCTGTAC GGAAGTGTTA CTTCTGCTCT 1150 AAAAGCTGCGGAATTGTACC CGCGGCCAGG AAAG ATG GTT CCT CAG 1196 Met Val Pro Gln 1 GCTCTC CTG TTT GTA CCC CTT CTG GTT TTT CCA TTG TGT TTT 1238 Ala Leu Leu PheVal Pro Leu Leu Val Phe Pro Leu Cys Phe 5 10 15 GGG AAA TTC CCT ATT TACACG ATA CTA GAC AAG CTT GGT CCC 1280 Gly Lys Phe Pro Ile Tyr Thr Ile LeuAsp Lys Leu Gly Pro 20 25 30 TGG AGC CCG ATT GAC ATA CAT CAC CTC AGC TGCCCA AAC AAT 1322 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn35 40 45 TTG GTA GTG GAG GAC GAA GGA TGC ACC AAC CTG TCA GGG TTC 1364Leu Val Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe 50 55 60 TCC TACATG GAA CTT AAA GTT GGA TAC ATC TTA GCC ATA AAA 1406 Ser Tyr Met Glu LeuLys Val Gly Tyr Ile Leu Ala Ile Lys 65 70 ATG AAC GGG TTC ACT TGC ACAGGC GTT GTG ACG GAG GCT GAA 1448 Met Asn Gly Phe Thr Cys Thr Gly Val ValThr Glu Ala Glu 75 80 85 ACC TAC ACT AAC TTC GTT GGT TAT GTC ACA ACC ACGTTC AAA 1490 Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr Thr Phe Lys 9095 100 AGA AAG CAT TTC CGC CCA ACA CCA GAT GCA TGT AGA GCC GCG 1532 ArgLys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala Ala 105 110 115 TAC AACTGG AAG ATG GCC GGT GAC CCC AGA TAT GAA GAG TCT 1574 Tyr Asn Trp Lys MetAla Gly Asp Pro Arg Tyr Glu Glu Ser 120 125 130 CTA CAC AAT CCG TAC CCTGAC TAC CGC TGG CTT CGA ACT GTA 1616 Leu His Asn Pro Tyr Pro Asp Tyr ArgTrp Leu Arg Thr Val 135 140 AAA ACC ACC AAG GAG TCT CTC GTT ATC ATA TCTCCA AGT GTA 1658 Lys Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val145 150 155 GCA GAT TTG GAC CCA TAT GAC AGA TCC CTT CAC TCG AGG GTC 1700Ala Asp Leu Asp Pro Tyr Asp Arg Ser Leu His Ser Arg Val 160 165 170 TTCCCT AGC GGG AAG TGC TCA GGA GTA GCG GTG TCT TCT ACC 1742 Phe Pro Ser GlyLys Cys Ser Gly Val Ala Val Ser Ser Thr 175 180 185 TAC TGC TCC ACT AACCAC GAT TAC ACC ATT TGG ATG CCC GAG 1784 Tyr Cys Ser Thr Asn His Asp TyrThr Ile Trp Met Pro Glu 190 195 200 AAT CCG AGA CTA GGG ATG TCT TGT GACATT TTT ACC AAT AGT 1826 Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe ThrAsn Ser 205 210 AGA GGG AAG AGA GCA TCC AAA GGG AGT GAG ACT TGC GGC TTT1868 Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly Phe 215 220 225GTA GAT GAA AGA GGC CTA TAT AAG TCT TTA AAA GGA GCA TGC 1910 Val Asp GluArg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys 230 235 240 AAA CTC AAG TTATGT GGA GTT CTA GGA CTT AGA CTT ATG GAT 1952 Lys Leu Lys Leu Cys Gly ValLeu Gly Leu Arg Leu Met Asp 245 250 255 GGA ACA TGG GTC GCG ATG CAA ACATCA AAT GAA ACC AAA TGG 1994 Gly Thr Trp Val Ala Met Gln Thr Ser Asn GluThr Lys Trp 260 265 270 TGC CCT CCC GAT CAG TTG GTG AAC CTG CAC GAC TTTCGC TCA 2036 Cys Pro Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser 275280 GAC GAA ATT GAG CAC CTT GTT GTA GAG GAG TTG GTC AGG AAG 2078 Asp GluIle Glu His Leu Val Val Glu Glu Leu Val Arg Lys 285 290 295 AGA GAG GAGTGT CTG GAT GCA CTA GAG TCC ATC ATG ACA ACC 2120 Arg Glu Glu Cys Leu AspAla Leu Glu Ser Ile Met Thr Thr 300 305 310 AAG TCA GTG AGT TTC AGA CGTCTC AGT CAT TTA AGA AAA CTT 2162 Lys Ser Val Ser Phe Arg Arg Leu Ser HisLeu Arg Lys Leu 315 320 325 GTC CCT GGG TTT GGA AAA GCA TAT ACC ATA TTCAAC AAG ACC 2204 Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys Thr330 335 340 TTG ATG GAA GCC GAT GCT CAC TAC AAG TCA GTC AGA ACT TGG 2246Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp 345 350 AAT GAGATC CTC CCT TCA AAA GGG TGT TTA AGA GTT GGG GGG 2288 Asn Glu Ile Leu ProSer Lys Gly Cys Leu Arg Val Gly Gly 355 360 365 AGG TGT CAT CCT CAT GTGAAC GGG GTG TTT TTC AAT GGT ATA 2330 Arg Cys His Pro His Val Asn Gly ValPhe Phe Asn Gly Ile 370 375 380 ATA TTA GGA CCT GAC GGC AAT GTC TTA ATCCCA GAG ATG CAA 2372 Ile Leu Gly Pro Asp Gly Asn Val Leu Ile Pro Glu MetGln 385 390 395 TCA TCC CTC CTC CAG CAA CAT ATG GAG TTG TTG GAA TCC TCG2414 Ser Ser Leu Leu Gln Gln His Met Glu Leu Leu Glu Ser Ser 400 405 410GTT ATC CCC CTT GTG CAC CCC CTG GCA GAC CCG TCT ACC GTT 2456 Val Ile ProLeu Val His Pro Leu Ala Asp Pro Ser Thr Val 415 420 TTC AAG GAC GGT GACGAG GCT GAG GAT TTT GTT GAA GTT CAC 2498 Phe Lys Asp Gly Asp Glu Ala GluAsp Phe Val Glu Val His 425 430 435 CTT CCC GAT GTG CAC AAT CAG GTC TCAGGA GTT GAC TTG GGT 2540 Leu Pro Asp Val His Asn Gln Val Ser Gly Val AspLeu Gly 440 445 450 CTC CCG AAC TGG GGG AAG TAT GTA TTA CTG AGT GCA GGGGCC 2582 Leu Pro Asn Trp Gly Lys Tyr Val Leu Leu Ser Ala Gly Ala 455 460465 CTG ACT GCC TTG ATG TTG ATA ATT TTC CTG ATG ACA TGT TGT 2624 Leu ThrAla Leu Met Leu Ile Ile Phe Leu Met Thr Cys Cys 470 475 480 AGA AGA GTCAAT CGA TCA GAA CCT ACG CAA CAC AAT CTC AGA 2666 Arg Arg Val Asn Arg SerGlu Pro Thr Gln His Asn Leu Arg 485 490 GGG ACA GGG AGG GAG GTG TCA GTCACT CCC CAA AGC GGG AAG 2708 Gly Thr Gly Arg Glu Val Ser Val Thr Pro GlnSer Gly Lys 495 500 505 ATC ATA TCT TCA TGG GAA TCA CAC AAG AGT GGG GGTGAG ACC 2750 Ile Ile Ser Ser Trp Glu Ser His Lys Ser Gly Gly Glu Thr 510515 520 AGA CTG TGAGGACTGG CCGTCCTTTC AACGATCCAA GTCCTGAAGA 2796 Arg LeuTCACCTCCCC TTGGGGGGTT CTTTTTAAAA AGGCCGCGGG GATCCAGACA 2846 TGATAAGATACATTGATGAG TTTGGACAAA CCACAACTAG AATGCAGTGA 2896 AAAAAATGCT TTATTTGTGAAATTTGTGAT GCTATTGCTT TATTTGTAAC 2946 CATTATAAGC TGCAATAAAC AAGTTAACAACAACAATTGC ATTCATTTTA 2996 TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTCGGATCCTCTAGAG 3046 TCGACCTGCA GGCTGATCTG GAAGGTGCTG AGGTACGATG AGACCCGCAC3096 CAGGTGCAGA CCCTGCGAGT GTGGCGGTAA ACATATTAGG AACCAGCCTG 3146TGATGCTGGA TGTGACCGAG GAGCTGAGGC CCGATCACTT GGTGCTGGCC 3196 TGCACCCGCGCTGAGTTTGG CTCTAGCGAT GAAGATACAG ATTGAGGTAC 3246 TGAAATGTGT GGGCGTGGCTTAAGGGTGGG AAAGAATATA TAAGGTGGGG 3296 GTCTTATGTA GTTTTGTATC TGTTTTGCAGCAGCCGCCGC CGCCATGAGC 3346 ACCAACTCGT TTGATGGAAG CATTGTGAGC TCATATTTGACAACGCGCAT 3396 GCCCCCATGG GCCGGGGTGC GTCAGAATGT GATGGGCTCC AGCATTGATG3446 GTCGCCCCGT CCTGCCCGCA AACTCTACTA CCTTGACCTA CGAGACCGTG 3496TCTGGAACGC CGTTGGAGAC TGCAGCCTCC GCCGCCGCTT CAGCCGCTGC 3546 AGCCACCGCCCGCGGGATTG TGACTGACTT TGCTTTCCTG AGCCCGCTTG 3596 CAAGCAGTGC AGCTTCCCGTTCATCCGCCC GCGATGACAA GTTGACGGCT 3646 CTTTTGGCAC AATTGGATTC TTTGACCCGGGAACTTAATG TCGTTTCTCA 3696 GCAGCTGTTG GATCTGCGCC AGCAGGTTTC TGCCCTGAAGGCTTCCTCCC 3746 CTCCCAATGC GGTTTAAAAC ATAAATAAAA AACCAGACTC TGTTTGGATT3796 TGGATCAAGC AAGTGTCTTG CTGTCTTTAT TTAGGGGTTT TGCGCGCGCG 3846GTAGGCCCGG GACCAGCGGT CTCGGTCGTT GAGGGTCCTG TGTATTTTTT 3896 CCAGGACGTGGTAAAGGTGA CTCTGGATGT TCAGATACAT GGGCATAAGC 3946 CCGTCTCTGG GGTGGAGGTAGCACCACTGC AGAGCTTCAT GCTGCGGGGT 3996 GGTGTTGTAG ATGATCCAGT CGTAGCAGGAGCGCTGGGCG TGGTGCCTAA 4046 AAATGTCTTT CAGTAGCAAG CTGATTGCCA GGGGCAGGCCCTTGGTGTAA 4096 GTGTTTACAA AGCGGTTAAG CTGGGATGGG TGCATACGTG GGGATATGAG4146 ATGCATCTTG GACTGTATTT TTAGGTTGGC TATGTTCCCA GCCATATCCC 4196TCCGGGGATT CATGTTGTGC AGAACCACCA GCACAGTGTA TCCGGTGCAC 4246 TTGGGAAATTTGTCATGTAG CTTAGAAGGA AATGCGTGGA AGAACTTGGA 4296 GACGCCCTTG TGACCTCCAAGATTTTCCAT GCATTCGTCC ATAATGATGG 4346 CAATGGGCCC ACGGGCGGCG GCCTGGGCGAAGATATTTCT GGGATCACTA 4396 ACGTCATAGT TGTGTTCCAG GATGAGATCG TCATAGGCCATTTTTACAAA 4446 GCGCGGGCGG AGGGTGCCAG ACTGCGGTAT AATGGTTCCA TCCGGCCCAG4496 GGGCGTAGTT ACCCTCACAG ATTTGCATTT CCCACGCTTT GAGTTCAGAT 4546GGGGGGATCA TGTCTACCTG CGGGGCGATG AAGAAAACGG TTTCCGGGGT 4596 AGGGGAGATCAGCTGGGAAG AAAGCAGGTT CCTGAGCAGC TGCGACTTAC 4646 CGCAGCCGGT GGGCCCGTAAATCACACCTA TTACCGGGTG CAACTGGTAG 4696 TTAAGAGAGC TGCAGCTGCC GTCATCCCTGAGCAGGGGGG CCACTTCGTT 4746 AAGCATGTCC CTGACTCGCA TGTTTTCCCT GACCAAATCCGCCAGAAGGC 4796 GCTCGCCGCC CAGCGATAGC AGTTCTTGCA AGGAAGCAAA GTTTTTCAAC4846 GGTTTGAGAC CGTCCGCCGT AGGCATGCTT TTGAGCGTTT GACCAAGCAG 4896TTCCAGGCGG TCCCACAGCT CGGTCACCTG CTCTACGGCA TCTCGATCCA 4946 GCATATCTCCTCGTTTCGCG GGTTGGGGCG GCTTTCGCTG TACGGCAGTA 4996 GTCGGTGCTC GTCCAGACGGGCCAGGGTCA TGTCTTTCCA CGGGCGCAGG 5046 GTCCTCGTCA GCGTAGTCTG GGTCACGGTGAAGGGGTGCG CTCCGGGCTG 5096 CGCGCTGGCC AGGGTGCGCT TGAGGCTGGT CCTGCTGGTGCTGAAGCGCT 5146 GCCGGTCTTC GCCCTGCGCG TCGGCCAGGT AGCATTTGAC CATGGTGTCA5196 TAGTCCAGCC CCTCCGCGGC GTGGCCCTTG GCGCGCAGCT TGCCCTTGGA 5246GGAGGCGCCG CACGAGGGGC AGTGCAGACT TTTGAGGGCG TAGAGCTTGG 5296 GCGCGAGAAATACCGATTCC GGGGAGTAGG CATCCGCGCC GCAGGCCCCG 5346 CAGACGGTCT CGCATTCCACGAGCCAGGTG AGCTCTGGCC GTTCGGGGTC 5396 AAAAACCAGG TTTCCCCCAT GCTTTTTGATGCGTTTCTTA CCTCTGGTTT 5446 CCATGAGCCG GTGTCCACGC TCGGTGACGA AAAGGCTGTCCGTGTCCCCG 5496 TATACAGACT TGAGAGGCCT GTCCTCGACC GATGCCCTTG AGAGCCTTCA5546 ACCCAGTCAG CTCCTTCCGG TGGGCGCGGG GCATGACTAT CGTCGCCGCA 5596CTTATGACTG TCTTCTTTAT CATGCAACTC GTAGGACAGG TGCCGGCAGC 5646 GCTCTGGGTCATTTTCGGCG AGGACCGCTT TCGCTGGAGC GCGACGATGA 5696 TCGGCCTGTC GCTTGCGGTATTCGGAATCT TGCACGCCCT CGCTCAAGCC 5746 TTCGTCACTG GTCCCGCCAC CAAACGTTTCGGCGAGAAGC AGGCCATTAT 5796 CGCCGGCATG GCGGCCGACG CGCTGGGCTA CGTCTTGCTGGCGTTCGCGA 5846 CGCGAGGCTG GATGGCCTTC CCCATTATGA TTCTTCTCGC TTCCGGCGGC5896 ATCGGGATGC CCGCGTTGCA GGCCATGCTG TCCAGGCAGG TAGATGACGA 5946CCATCAGGGA CAGCTTCAAG GATCGCTCGC GGCTCTTACC AGCCTAACTT 5996 CGATCACTGGACCGCTGATC GTCACGGCGA TTTATGCCGC CTCGGCGAGC 6046 ACATGGAACG GGTTGGCATGGATTGTAGGC GCCGCCCTAT ACCTTGTCTG 6096 CCTCCCCGCG TTGCGTCGCG GTGCATGGAGCCGGGCCACC TCGACCTGAA 6146 TGGAAGCCGG CGGCACCTCG CTAACGGATT CACCACTCCAAGAATTGGAG 6196 CCAATCAATT CTTGCGGAGA ACTGTGAATG CGCAAACCAA CCCTTGGCAG6246 AACATATCCA TCGCGTCCGC CATCTCCAGC AGCCGCACGC GGCGCATCTC 6296GGGCAGCGTT GGGTCCTGGC CACGGGTGCG CATGATCGTG CTCCTGTCGT 6346 TGAGGACCCGGCTAGGCTGG CGGGGTTGCC TTACTGGTTA GCAGAATGAA 6396 TCACCGATAC GCGAGCGAACGTGAAGCGAC TGCTGCTGCA AAACGTCTGC 6446 GACCTGAGCA ACAACATGAA TGGTCTTCGGTTTCCGTGTT TCGTAAAGTC 6496 TGGAAACGCG GAAGTCAGCG CCCTGCACCA TTATGTTCCGGATCTGCATC 6546 GCAGGATGCT GCTGGCTACC CTGTGGAACA CCTACATCTG TATTAACGAA6596 GCCTTTCTCA ATGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT 6646CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG 6696 CGCCTTATCCGGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT 6746 TATCGCCACT GGCAGCAGCCACTGGTAACA GGATTAGCAG AGCGAGGTAT 6796 GTAGGCGGTG CTACAGAGTT CTTGAAGTGGTGGCCTAACT ACGGCTACAC 6846 TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCAGTTACCTTCG 6896 GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC6946 GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC 6996TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG 7046 AAAACTCACGTTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC 7096 ACCTAGATCC TTTTAAATTAAAAATGAAGT TTTAAATCAA TCTAAAGTAT 7146 ATATGAGTAA ACTTGGTCTG ACAGTTACCAATGCTTAATC AGTGAGGCAC 7196 CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGCCTGACTCCCC 7246 GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC7296 TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA 7346TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA 7396 TCCGCCTCCATCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG 7446 TTCGCCAGTT AATAGTTTGCGCAACGTTGT TGCCATTGCT GCAGGCATCG 7496 TGGTGTCACG CTCGTCGTTT GGTATGGCTTCATTCAGCTC CGGTTCCCAA 7546 CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAAAAGCGGTTAG 7596 CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT7646 CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC 7696GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA 7746 ATAGTGTATGCGGCGACCGA GTTGCTCTTG CCCGGCGTCA ACACGGGATA 7796 ATACCGCGCC ACATAGCAGAACTTTAAAAG TGCTCATCAT TGGAAAACGT 7846 TCTTCGGGGC GAAAACTCTC AAGGATCTTACCGCTGTTGA GATCCAGTTC 7896 GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCTTTTACTTTCA 7946 CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG7996 GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA 8046ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT 8096 TTGAATGTATTTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC 8146 CGAAAAGTGC CACCTGACGTCTAAGAAACC ATTATTATCA TGACATTAAC 8196 CTATAAAAAT AGGCGTATCA CGAGGCCCTTTCGTCTTCAA 8236 524 amino acids amino acid linear protein not provided 2Met Val Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro 1 5 10 15Leu Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Leu Asp Lys Leu 20 25 30 GlyPro Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn 35 40 45 Asn LeuVal Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe 50 55 60 Ser Tyr MetGlu Leu Lys Val Gly Tyr Ile Leu Ala Ile Lys Met 65 70 75 Asn Gly Phe ThrCys Thr Gly Val Val Thr Glu Ala Glu Thr Tyr 80 85 90 Thr Asn Phe Val GlyTyr Val Thr Thr Thr Phe Lys Arg Lys His 95 100 105 Phe Arg Pro Thr ProAsp Ala Cys Arg Ala Ala Tyr Asn Trp Lys 110 115 120 Met Ala Gly Asp ProArg Tyr Glu Glu Ser Leu His Asn Pro Tyr 125 130 135 Pro Asp Tyr Arg TrpLeu Arg Thr Val Lys Thr Thr Lys Glu Ser 140 145 150 Leu Val Ile Ile SerPro Ser Val Ala Asp Leu Asp Pro Tyr Asp 155 160 165 Arg Ser Leu His SerArg Val Phe Pro Ser Gly Lys Cys Ser Gly 170 175 180 Val Ala Val Ser SerThr Tyr Cys Ser Thr Asn His Asp Tyr Thr 185 190 195 Ile Trp Met Pro GluAsn Pro Arg Leu Gly Met Ser Cys Asp Ile 200 205 210 Phe Thr Asn Ser ArgGly Lys Arg Ala Ser Lys Gly Ser Glu Thr 215 220 225 Cys Gly Phe Val AspGlu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 230 235 240 Ala Cys Lys Leu LysLeu Cys Gly Val Leu Gly Leu Arg Leu Met 245 250 255 Asp Gly Thr Trp ValAla Met Gln Thr Ser Asn Glu Thr Lys Trp 260 265 270 Cys Pro Pro Asp GlnLeu Val Asn Leu His Asp Phe Arg Ser Asp 275 280 285 Glu Ile Glu His LeuVal Val Glu Glu Leu Val Arg Lys Arg Glu 290 295 300 Glu Cys Leu Asp AlaLeu Glu Ser Ile Met Thr Thr Lys Ser Val 305 310 315 Ser Phe Arg Arg LeuSer His Leu Arg Lys Leu Val Pro Gly Phe 320 325 330 Gly Lys Ala Tyr ThrIle Phe Asn Lys Thr Leu Met Glu Ala Asp 335 340 345 Ala His Tyr Lys SerVal Arg Thr Trp Asn Glu Ile Leu Pro Ser 350 355 360 Lys Gly Cys Leu ArgVal Gly Gly Arg Cys His Pro His Val Asn 365 370 375 Gly Val Phe Phe AsnGly Ile Ile Leu Gly Pro Asp Gly Asn Val 380 385 390 Leu Ile Pro Glu MetGln Ser Ser Leu Leu Gln Gln His Met Glu 395 400 405 Leu Leu Glu Ser SerVal Ile Pro Leu Val His Pro Leu Ala Asp 410 415 420 Pro Ser Thr Val PheLys Asp Gly Asp Glu Ala Glu Asp Phe Val 425 430 435 Glu Val His Leu ProAsp Val His Asn Gln Val Ser Gly Val Asp 440 445 450 Leu Gly Leu Pro AsnTrp Gly Lys Tyr Val Leu Leu Ser Ala Gly 455 460 465 Ala Leu Thr Ala LeuMet Leu Ile Ile Phe Leu Met Thr Cys Cys 470 475 480 Arg Arg Val Asn ArgSer Glu Pro Thr Gln His Asn Leu Arg Gly 485 490 495 Thr Gly Arg Glu ValSer Val Thr Pro Gln Ser Gly Lys Ile Ile 500 505 510 Ser Ser Trp Glu SerHis Lys Ser Gly Gly Glu Thr Arg Leu 515 520

What is claimed is:
 1. A method of inducing an immune response in amammal against human immunodeficiency virus HIV comprising:administering to said mammal a sufficient amount of a recombinantadenovirus comprising a human serotype 5 adenovirus containing acomplete deletion of its E1 gene and at least a functional deletion ofits E3 gene, and, in the site of the E1 gene deletion, a sequencecomprising a cytomegalovirus promoter directing the expression of DNAencoding an HIV protein, which when administered to the mammal in saidrecombinant adenovirus, elicits an immune response against HIV.
 2. Themethod according to claim 1, wherein said HIV protein is an HIV gp120protein.
 3. The method according to claim 1 wherein said adenovirus isadministered subcutaneously, intranasally, intratracheally, orintramuscularly.
 4. A method of inducing an immune response in a mammalagainst a selected pathogen comprising: administering to said human asufficient amount of a recombinant adenovirus comprising a humanserotype 5 adenovirus containing a complete deletion of its E1 gene andat least a functional deletion of its E3 gene from adenovirus map units78.5-84.3, and, in the site of the E1 gene deletion, a sequencecomprising a cytomegalovirus promoter directing the expression of DNAencoding a protein of said pathogen, which when administered to thehuman in said recombinant virus, elicits an immune response against saidpathogen.